2. Introduction
Supercritical fluid chromatography (SFC) employs, as its name
suggests, a fluid in the supercritical state as its mobile phase.
This leads to improvements in the separations of thermo labile
compounds and more generally for compounds of high molecular
weight. The instrument is conceptually a hybrid of gas
chromatograph and liquid chromatograph, with either GC capillary
columns or HPLC columns, the latter being preferred.
Supercritical Fluid Chromatography is a form of normal phase
chromatography. It can also be used for the separation of Chiral
compounds. Principles are similar to those of high performance
liquid chromatography (HPLC), however SFC typically utilizes
carbon dioxide as the mobile phase; therefore the entire
chromatographic flow path must be pressurized.
3. Compared to gas chromatography, where gas is under
ambient pressure, and liquid chromatography, where liquid
is used as mobile phase, the solvent power of the fluid
mobile phase in SFC can be varied by density, e.g., by
pressure changes at constant temperature. Solubility
generally increases with pressure under supercritical
conditions of the mobile phase.
Since temperature is near the critical temperature of the
mobile phase, temperature sensitive compounds can be
processed. Chromatographic separation can be carried out
at constant pressure (isobaric operation) or with increasing
pressure (pressure programmed). In addition, temperature
can be varied.
4. Temperature directly determines the vapor pressure of
the feed components and the density of the mobile
phase and, indirectly, adsorption equilibrium. With
higher temperature, vapor pressures of the feed
components increase exponentially.
Density decreases proportionally to temperature if
conditions are far from critical, but in the region of the
critical point of the mobile phase, which is the main
area of application of supercritical fluid
chromatography, density varies dramatically with
temperature. In this region, the solvent power of the
mobile phase, which increases with density, is
substantially changed.
5. Supercritical Fluids
The transformation of a pure compound from a liquid to a gaseous state
and vice versa corresponds to a phase change that can be induced over a
limited domain by pressure or temperature. For example, a pure substance
in the gaseous state cannot be liquefied above a given temperature, called
the critical temperature Tc, irrespective of the pressure applied to it. The
minimum pressure required to liquefy a gas at its critical temperature is
called the critical pressure Pc.
These points are the defining boundaries on a phase diagram for a pure
substance. The curve, which limits the gas and liquid domains, stops at the
critical point C. Under these conditions, gas and liquid states have the
same density. Above these temperatures and pressures, the compound
becomes a supercritical fluid. In particular the viscosity of a supercritical
fluid is almost that of a gas
The use of a supercritical fluid mobile phase in chromatography was first
proposed in 1958 by J. Lovelock. The first actual report use of this in a
chromatographic system was in 1962 by Klesper et al, who used it to
separate thermally-labile porphyrins.
6. A pure supercritical fluid (SCF) is any compound at a
temperature and pressure above the critical values (above
critical point).
The fluid, as it is termed, is neither a gas nor a liquid and
is best described as intermediate to the two extremes.
This phase retains solvent power approximating liquids
as well as the transport properties common to gases.
Supercritical fluid, as its called, is heavy like liquid but
with penetration power of gas. These qualities make
supercritical fluids effective and selective solvents.
7.
8. Property Density (kg/m3 ) Viscosity (cP) Diffusivity (mm2 /s)
Gas 1 0.01 1-10
SCF 100-800 0.05-0.1 0.01-0.1
Liquid 1000 0.5-1.0 0.001
A comparison of typical values for density, viscosity and diffusivity of gases,
liquids, and SCFs
9. Some of the advantages and disadvantages of SCFs compared to conventional
liquid solvents for separations:
Advantages
• Dissolving power of the SCF is controlled by pressure and/or temperature
• SCF is easily recoverable from the extract due to its volatility
• Non-toxic solvents leave no harmful residue
• High boiling components are extracted at relatively low temperatures
• Separations not possible by more traditional processes can sometimes be effected
• Thermally labile compounds can be extracted with minimal damage as low
temperatures can be employed by the extraction
Disadvantages
• Elevated pressure required
• Compression of solvent requires elaborate recycling measures to reduce energy
costs
• High capital investment for equipment
10. 10
What Compounds Can SFC
Separate?
Any solute soluble in methanol or a less polar
organic solvent will elute in SFC.
Strong organic acids and bases require a modifier
and an additive in the mobile phase.
Most salts of organic acids and bases elute.
Small lipophylic peptides elute
With carbon dioxide based fluids, molecular
weights up to 15,000
11. 11
What Compounds Will NOT Elute
in SFC?
Any solutes requiring an aqueous environment
any solutes requiring a buffered or ionic aqueous
environment
I.E., Biomolecules such as Proteins
will NOT elute
inorganic salts will not elute
With carbon dioxide, few solutes over 15,000 MW
elute
12. SFC Advantages vs HPLC
Supercritical fluids have low viscosities
- faster analysis (5 to 10 X faster)
- less pressure drop across the column
- the use of open tubular columns is feasible
Column lengths from 10 to 20 m and inside
diameters of 50 or 100 m are used
Can be used with a wide range of sensitive
detectors
Resolving power is ~5X that of HPLC
13. SFC Advantages vs GC
Can analyze non-volatile, polar, or adsorptive
solutes without derivatization.
Can analyze thermally labile compounds.
Can analyze solutes of much higher molecular
weight.
17. Mobile Phases
Non-polar or low-polarity compounds have been explored
for use as mobile phases in SFC.
These include: CO2, N2O, SF6, xenon, ethane, propane,
pentane
Separations are a combination of low polarity mobile
phases with relatively polar stationary phases, so SFC
typically functions in the normal-phase mode.
Elution is a function of molecular mass and polarity. Larger
or more polar compounds have longer retention times.
Most common mobile phase is carbon dioxide (with and
without polar additive).
18. Carbon dioxide is nontoxic, nonflammable, noncorrosive, available in
high purity, low cost, inert to most substances, and does not interfere
with most detection methods.
Under normal SFC operating conditions, CO2 has a wide density range
which provides optimum solubilizing power.
CO2 must be 99.9995% pure for FID (no hydrocarbons) and 99.8% pure for
UV.
Small amounts of a modifier (organic solvent) can be added to the
supercritical fluid. Most common are methanol and water.
This results in a change in solvent polarity and nature and follows Snyder
rules.
19. Why Carbon Dioxide?
Miscible with much more polar solvents-wide range of
solvent strength available.
program composition from 0 to 100% organic modifier
Low operating temperatures (i.e., 30-50°C) Compatible
with most HPLC and GC Detectors
Relatively safe
product of human respiration
can use with the FID
easy disposal
low purchase price
Lack of a superior alternative
19
20. Instrumentation - Pump
− Functions to maintain a suitable, precise mobile phase flow, and used to apply
pressure to keep the mobile phase in the supercritical state.
− Both pressure and temperature must be precisely controlled so use an oven
also.
− Mobile phases enter the pump as a liquid from a cylinder and is pumped to the
column where it is heated to the supercritical state.
− On-line filters and activated carbon or alumina adsorption cartridges are
employed to purify the mobile phase prior to entering the pump.
− Pump design and seals are selected to tolerate very high solvent strengths
and pressures.
− Typically syringe pumps or reciprocating pumps are used for 1ul/min to 10
ml/min flow rates.
− Syringe pumps deliver pulseless flow, but have a fixed delivery volume and
require a refilling cycle.
− Reciprocating pumps need no refilling but require a pressure dampening
device.
21. Instrumentation - Pressure Programming
Similar to temperature programming for GC or gradient elution for
HPLC.
As pressure increases, so does the density – method may be called
density programming.
Density programming results in increasing the solubility of the
solutes in the mobile phase.
As P increases, retention decreases.
Two types of pressure programming:
• linear – pressure is increased at a fixed rate
• asymptotic – rate is decreased linearly as it approaches a
maximum
22.
23. Injectors
Typical HPLC design injectors for packed
columns.
Split/Splitless valve injector (0.01 to 0.05
L injections) for open tubular columns.
Timed - split injector (0.01 to 0.05 L
injections) for open tubular columns.
24. Injection Systems
Sample introduction is based on the high-pressure
rotary valve system used for HPLC.
Problems with injection devices for SCF include;
• peak distortion and loss of resolution
• discrimination and memory effects
• poor reproducibility
• lack of concentration sensitivity
To overcome these problems several approaches have
been used, including;
• splitting devices
• solvent venting
25. Split/Splitless Valve
Injector
Similar to GC type also called
dynamic split system.
When valve is in sampling position,
the sample is injected under ambient
conditions into the sample loop.
When valve is switched to load
position, sample is forced into the
splitter by flow of high pressure
mobile phase.
Split vent is open and sample is split
with a small fraction entering the
column.
Splitless mode is when the split vent
valve is kept closed.
26. Timed-Split Injector
Splitting of sample is done
by quickly switching back
and forth between load to
inject position so that only
a fraction of the sample
enters the column.
Electronic or pneumatic
actuators can move the
valve as fast as 10 ms
injecting 1-2 nL of sample.
Reproducibility is ~ 4%.
27. Columns
Two types of columns used;
- Open tubular
- Packed
Open tubular is preferred because pressure control
is easier.
28. Columns
Open tubular columns for SFC have smaller diameters
than those used in GC.
However, column preparation and stationary phases
are the same as used in GC.
Polysiloxanes are the most popular stationary phases,
since they have high thermal stability and low
viscosity.
Polarity is varied by incorporating different functional
groups into the polymer and cross-linking is essential
to ensure stability.
29. Restrictors
Placed between the column and detector or after the
detector to maintain fluid flow and supercritical fluid
conditions along the length of the column but affect
rapid decompression to atmospheric pressure before
detection.
The difficulty is transferring the eluent from the
supercritical phase to the gas phase without
compromising column efficiency by introducing
excessive dead volume or causing sample components
to condense.
30. Ideal restrictors do not exist but need to have some of
the following properties:
• inert
• immune from plugging
• adjustable
• easily replaced
• effective for all samples
Linear - Initial restrictors were short lengths of narrow-
bore fused silica connected to the column end.
Generate a linear pressure gradient and work well for
low molecular mass analytes and gas phase detectors.
Tapered and integral – restrictors which help with rapid
decompression over a short path length. Tip is heated
to avoid condensation of analyte, better for polar and
high molecular mass analytes.
31.
32. 32
Detectors
•FID detection
•Coupling of mass spectrometry is considerably easier to achieve
by comparisons to LC and has been used.
•UV, IR, Fluorescence, Flame photometric, TCD and ECD can also
be employed.
- DAD for purity assessment
- MS to verify presence of target
- NCD to quantify on nitrogen content
- FTIR
33. Factors affecting retention
Dependent on temperature, pressure, mobile phase
density, and composition of the stationary and mobile
phases.
Supercritical fluids most important properties are
viscosity, diffusivity, and solvating power.
Solvating power relates to the capacity of the
supercritical fluid for specific intermolecular interactions.
Solvating power for solvents is expressed by the
Hildebrand solubility parameter, d.
34. 34
1- The density of CO2 and solvent power decreases as the temperature
increases, which results in an increase in k.
2- The vapor pressure of the analyte also increases at higher temperature,
leading to an increase in solubility and thus a decrease in k.
•The second effect over compensates the first around 100oC(1/T=0.0028),
resulting in a maximum in retention factor.
•At temperatures higher than 100oC , the second effect becomes more
pronounced and k values decrease with increasing temperature.
•At high pressure the decrease in density with increasing temperature is not as
important as the effect of temperature on solvating power. Therefore plots of
k vs. 1/T are usually more flat. The two variables effects may compensate
each other.
Effect of temperature on retention factor
35. 35
Effect of temperature on efficiency
Efficiency generally increases with an increase in temperature
1) Mobile phase diffusivity increase with temperature at a constant
pressure and increases N.
2) Decreased density at higher temperatures lead to a drop in mobile
phase viscosity which increases N.
3) Retention factor increases which will increase N.
However it has been seen that efficiency has increased with
temperature to a point and then levels off. Then as the critical
temperature is transverse column efficiency declines. This is
attributed to the adsorption of solvent to the stationary phase. The
presence of this adsorbed layer would slow the kinetics of binding
and hence a decline in column efficiency.
36. 36
s
R
1
1
4 2
2
k
k
N
Resolution depends on column efficiency, solute retention and
selectivity.
In SFC we can vary the variables to obtain the desired
resolution.
Ex. Increase the temperature will
* Decrease the CO2 density
* May increase or decrease the retention factor
* Vapor pressure of the analyte also increases
* Increases rate of diffusion (increased efficiency)
Depending on what variable/variables predominate the Rs could
improve or not.
Effect of temperature on resolution
37. 37
Effect of pressure
Pressure has a little effect on the selectivity
Pressure affects linear velocity, efficiency, and retention factor.
Therefore resolution depends on the effective pressure.
Retention factor decreases with increasing pressure in SFC since
the density of CO2 and solvent power increases.
At increasing pressures mobile phases have greater viscosities and
greater mass transfer resistance which may lead to decreased
efficiency.
38. Supercritical Extraction
For over a quarter of a century supercritical fluids, primarily carbon dioxide, have been
used as a solvent in extraction processes performed under supercritical conditions.
Carbon Dioxide has several properties that recommend it for this duty. Its critical
temperature is 31.3C, making near room temperature operations possible. It is non-
toxic, non-flammable and approved by FDA for use in food and pharmaceutical plants.
Its critical pressure is 72.9 atm., which is considered moderate. Its properties have been
exhaustively studied, so it continues to be the extraction solvent of choice. Caffeine
removal is an excellent example of the application of this technology.
Most supercritical Fluid Extraction processes are quite simple . A sample is placed in
the Sample thimble, and supercritical fluid is pumped through the thimble. The
extraction of the soluble compounds is allowed to take place as the supercritical fluid
passes into a collection trap through a restricting nozzle. The fluid is vented in the
collection trap, allowing the solvent to either escape or be recompressed for future use.
The material left behind in the collection trap is the product of the extraction.
Obviously this is a batch process. This is acceptable for the analytical purpose to which
the method is applied, but could not be considered commercially, unless there was
some extreme purpose to which the process was being applied.
39. Solvents of supercritical fluid extraction
The choice of the SFE solvent is similar to the regular extraction. Principle
considerations are the followings.
• Good solvating property
• Inert to the product
• Easy separation from the product
• Cheap
• Low Purchase Cost because of economic reasons
Carbon dioxide is the most commonly used SCF, due primarily to its low critical
parameters (31.1°C, 72.8 bar), low cost and non-toxicity. Limited to nonpolar
solutes. Some polarity adjustment by adding 5-20% organic co solvents (called
“modifiers”). However, several other SCFs have been used in both commercial
and development processes.
42. Compound Tcº C Pc atm d*
CO2 31.3 72.9 0.96
C2H4 9.9 50.5 ---
N2O 36.5 72.5 0.94
NH3 132.5 112.5 0.40
n-C5 196.6 33.3 0.51
n-C4 152.0 37.5 0.50
CCl2F2 111.8 40.7 1.12
CHF3 25.9 46.9 ---
H2O 374.1 218.3 ---
Typical Supercritical Solvents
* Density in g/ml at 400 atm.
43. Capillary SFC
The recently introduced capillary columns seemed
ideal candidates on which to perform such
supercritical fluid chromatography (SFC) methods.
In addition, gas chromatography (GC) detectors,
including flame ionization, electron capture and
nitrogen phosphorous, were optimized for SFC
with additional heating zones to take into account
adiabatic cooling during the decompression phase.
In these early days, the targeted application areas
were primarily in the petrochemical industry. It is
interesting to note that most pioneers from the
pharmaceutical industry who tested the available
instruments found the technology very limited, if
not almost useless.
44. Packed column SFC
The company began by modifying the only available analytical
SFC instrument to produce the first modular packed-column
SFC system dedicated to pharmaceutical applications. This
instrument demonstrated resolution similar to, if not better than,
reversed-phase HPLC, and represented a rebirth of normal-phase
chromatography. By taking advantage of the very low viscosity
and high diffusivity of the mobile phase — CO2 and modifier
(usually methanol) — very fast column equilibration and higher
optimum flow-rates were possible, and very long columns (up to
8 × 25 cm) packed with smaller particles, often in super-
optimum flow-rates, could be used. The ability to use optimized
GC detectors was maintained and some SFC users even
developed interfaces (or specific cells) to collect simultaneous
data from multiple detectors in single runs, including diode
array, mass spectrometry, evaporative light scattering, atomic
emission, and Fourier transform infra red spectroscopy.
46. Industrial applications
Food and flavoring
SFE is applied in food and flavoring industry as the residual solvent could be easily removed
from the product no matter whether it is the extract or the extracted matrix. The biggest
application is the de caffeination of tea and coffee. Other important areas are the extraction of
essential oils and aroma materials from spices. Brewery industry uses SFE for the extraction of
hop. The method is used in extracting some edible oils and producing cholesterine-free egg
powder.
Petrol chemistry
The distillation residue of the crude oil is handled with SFE as a custom large-scale procedure
(ROSE Residuum Oil Supercritical Extraction). The method is applied in regeneration
procedures of used oils and lubricants.
Pharmaceutical industry
Producing of active ingredients from herbal plants for avoiding thermo or chemical degradation.
Elimination of residual solvents from the products.
Other plant extractions
Production of de nicotined tobacco.
Environmental protection
Elimination of residual solvents from wastes. Purification of contaminated soil.
47. SUPERCRITICAL FLUIDS:
NANOTECHNOLOGY APPLICATIONS
Nanoparticles: Synthesis Using SCF
As indicated above, although nanoparticles made of
different materials have different enhanced physical,
chemical, or optical properties, they are considered
as important building blocks in nanotechnology. For
this reason alone, research on methods for
synthesis of nanoparticles of different materials is
considered an important pillar of this new
technology. In what follows, only those methods that
employ special properties of SCFs are as follows:
48. Rapid expansion supercritical solution (RESS):
In early studies, Krukonis (1984) demonstrated an
application of the RESS method for particle synthesis.
Small particles and fibres of many materials including
aluminum isopropoxide, dodecanolactam,
polypropylene, b-estradiol, ferrocene, navy blue dye,
and soybean lecithin were produced by this method.
In the RESS process a solute is dissolved in a
supercritical fluid and then the solution is rapidly
expanded through a small nozzle or an orifice into a
space of lower pressure. The rapid reduction of
density (as a result of lowered pressure) to the
subcritical range causes rapid precipitation of the
solute and subsequent formation of nanoparticles.
49. The supercritical fluid is obtained by heating and pressurization
of the solution from room temperature, allowing the expansion
phase of the process to occur at a known concentration (as
opposed to continuously extracting the solute using an
extraction column).
Because solubilities in SCFs can be higher than those under
ideal gas condition by many orders of magnitude (about a
million times), rapid expansion from a supercritical pressure
results in extremely high supersaturation and consequently
homogeneous nucleation of the solute, forming a narrow size-
distribution of particles.
A variety of product morphologies, such as particles, films, and
fibres from organic, inorganic, and polymeric materials are
possible.
50. As an example, supercritical water is used as a solvent with chips of
high purity SiO2 glass in an autoclave as shown in Figure. The
autoclave at a subcritical temperature and pressure of 590 bar was
equilibrated for hours.
Then the valve was opened and a heated tube zone raised the
temperature to a supercritical value. Rapid expansion in an expansion
nozzle (5mm length, 60 mm i.d. s.s. tube) into an evacuated chamber
(0.1 to1 bar), to minimize potential health hazards, produced fine
powders of silica.
A dramatic change in the dissolving power experienced by a solute as it
rapidly expands from a supercritical state (significant dissolving power)
to a subcritical state (negligible dissolving power) started rapid
nucleation and growth processes of the low vapor pressure solute
leading to the formation of fine particles. The expansion process is
divided into three stages: subcritical expansion (through the length of
the nozzles itself), a brief supersonic free jet expansion (immediately
upon exiting the nozzle), and the final stage in which the jet interacts
significantly with the background gas in the expansion region.
51.
52. Supercritical antisolvent method (SAS): This method of micro-
and nano-particle synthesis uses the precipitation processes in
supercritical solutions known as Supercritical AntiSolvent (SAS). The
case of interest here is when a solution is added or injected into a
supercritical fluid acting as the antisolvent agent, see Figure. This
method requires that the antisolvent be miscible with the solvent in
the solution and that the solute be insoluble in the supercritical
antisolvent (this is referred to as reverseaddition precipitation).
Generally in this approach, a liquid solution is sprayed through a
nozzle or an orifice into a supercritical fluid antisolvent. The fast
diffusion of the solvent from the solution drops in the spray results in
solute precipitation. The precipitate is subsequently washed with the
antiosolvent and filtered to collect the particles.
An important advantage of the SAS and other supercritical methods
over liquid-solution based approaches is the fact that dry powders
are produced in a single step.
53.
54. For some applications, such as in pharmaceuticals and in protein
samples, synthesis at low temperatures is critical to prevent material
damage by thermal shock. Also, this method has generally higher
throughput than the RESS approach mainly because many compounds of
interest have higher solubility in liquid solvents than in low-temperature
supercritical fluids. This method has been employed in the production of a
variety of fine particles and powders from pharmaceuticals, pigments,
polymers, proteins, and explosive materials.
For example, for polymers, Mawsonet al. (1997) utilized a toluene solution
of polystyrene sprayed into compressed CO2. The particle size was
adjustable within a wide range (100 nm to 20 mm) by varying the CO2
density and temperature. Higher CO2 densities and lower temperatures
resulted in smaller particles.
Application to explosives has been demonstrated by Gallagher et al.
(1992). Very small crystalline particles (<200 mm) of
cyclotrimethylenetrinitramine (RDX) were made using subcritical and
supercritical CO2 as the antisolvent with a variety of solvents.
